Structural bias in T4 RNA ligase-mediated 3′-adapter ligation

Abstract

T4 RNA ligases are commonly used to attach adapters to RNAs, but large differences in ligation efficiency make detection and quantitation problematic. We developed a ligation selection strategy using random RNAs in combination with high-throughput sequencing to gain insight into the differences in efficiency of ligating pre-adenylated DNA adapters to RNA 3′-ends. After analyzing biases in RNA sequence, secondary structure and RNA-adapter cofold structure, we conclude that T4 RNA ligases do not show significant primary sequence preference in RNA substrates, but are biased against structural features within RNAs and adapters. Specifically, RNAs with less than three unstructured nucleotides at the 3′-end and RNAs that are predicted to cofold with an adapter in unfavorable structures are likely to be poorly ligated. The effect of RNA-adapter cofold structures on ligation is supported by experiments where the ligation efficiency of specific miRNAs was changed by designing adapters to alter cofold structure. In addition, we show that using adapters with randomized regions results in higher ligation efficiency and reduced ligation bias. We propose that using randomized adapters may improve RNA representation in experiments that include a 3′-adapter ligation step.

Lessons from microRNA Sequencing Using Illumina Technology

“…Our data confirmed the strong sequence and/or structural-fold preference of RNA ligases ultimately leading to 3'-or 5'-adaptor-dependent miRNA profiles [20]. To solve this problem and allow a more accurate measurement of the absolute expression level of miRNA, a strategy had been proposed using pools of adaptors degenerated at their first positions [8] [17] [18] [20]. However, this cannot be sufficient to achieve the "true" miRNome characterization as we reported that profiles recovered with a family of barcoded adaptors displayed variation far below the variation observed between adaptors differing over their entire length for a large number of miRNAs (personal data).…”

“…Four categories of miRNAs can be detected according to expression fold changes: 1 to 3, 3 to 10, 10 to 50, and beyond. Our data confirmed the strong sequence and/or structural-fold preference of RNA ligases ultimately leading to 3'-or 5'-adaptor-dependent miRNA profiles [20]. To solve this problem and allow a more accurate measurement of the absolute expression level of miRNA, a strategy had been proposed using pools of adaptors degenerated at their first positions [8] [17] [18] [20].…”

Whole-genome mapping of 5′ RNA ends in bacteria by tagged sequencing: a comprehensive view in Enterococcus faecalis

“…This effect may be further strengthened by spontaneous hydrolysis of 5 ′ triphosphate RNA ends during the ligation step of the PSS-adaptor and preceding RNA treatments, generating 5 ′ ends opened for ligation. On the other hand, the first step of the tagging procedure using the T4 RNA ligase is certainly not complete and acts with different efficiency on different RNA molecules (Zhuang et al 2012;Raabe et al 2014). Therefore, at the second ligation step, 5 ′ monophosphate ends (i.e., PSSs) that have escaped the first tag can be ligated to the TSS-adaptor and appear as false TSSs.…”

RNA-seq of human reference RNA samples using a thermostable group II intron reverse transcriptase

“…The Illumina Small RNA Kit uses adapters that target small ncRNAs, such as miRNAs and snoRNAs, with 5 ′ monophosphate and 3 ′ OH termini (Illumina product literature) followed by size-selection of cDNAs prior to sequencing. This and similar methods using sequential RNA-seq adapter ligation are typically time consuming, have well-documented sequence and secondary structure biases, and can yield undesirable side products (Hafner et al 2011;Zhuang et al 2012;Raabe et al 2014).…”